External control cohorts for the single-arm LIBRETTO-001 trial of selpercatinib in RET+ non-small-cell lung cancer.

BACKGROUND: Data for selpercatinib [a selective REarranged during Transfection (RET) inhibitor] from a single-arm trial (LIBRETTO-001, NCT03157128) in RET-fusion-positive advanced/metastatic non-small-cell lung cancer (NSCLC) were used in combination with external data sources to estimate comparative efficacy [objective response rate (ORR), progression-free survival, and overall survival (OS)] in first- and second-line treatment settings. METHODS: Patient-level data were obtained from a de-identified real-world database. Patients diagnosed with advanced/metastatic NSCLC with no prior exposure to a RET inhibitor and one or more prior line of therapy were eligible. Additionally, individual patient-level data (IPD) were obtained from the pemetrexed + platinum arm of KEYNOTE-189 (NCT03950674, first line) and the docetaxel arm of REVEL (NCT01168973, post-progression). Patients were matched using entropy balancing, doubly robust method, and propensity score approaches. For patients with unknown/negative RET status, adjustment was made using a model fitted to IPD from a real-world database. RESULTS: In first-line unadjusted analyses of the real-world control, ORR was 87.2% for LIBRETTO-001 versus 66.7% for those with RET-positive NSCLC (P = 0.06). After adjustment for unknown RET status and other patient characteristics, selpercatinib remained significantly superior versus the real-world control for all outcomes (all P < 0.001 except unadjusted RET-fusion-positive cohort). Similarly, outcomes were significantly improved versus clinical trial controls (all P < 0.05). CONCLUSIONS: Findings suggest improvement in outcomes associated with selpercatinib treatment versus the multiple external control cohorts, but should be interpreted with caution. Data were limited by the rarity of RET, lack of mature OS data, and uncertainty from assumptions to create control arms from external data.

The increased mucosal mRNA expressions of complement C3 and interleukin-17 in inflammatory bowel disease.

Recent studies have demonstrated that the complement system participates in the regulation of T cell functions. To address the local biosynthesis of complement components in inflammatory bowel disease (IBD) mucosa, we investigated C3 and interleukin (IL)-17 mRNA expression in mucosal samples obtained from patients with IBD. The molecular mechanisms underlying C3 induction were investigated in human colonic subepithelial myofibroblasts (SEMFs). IL-17 and C3 mRNA expressions in the IBD mucosa were evaluated by real-time polymerase chain reaction. The C3 levels in the supernatant were determined by enzyme-linked immunosorbent assay. IL-17 and C3 mRNA expressions were elevated significantly in the active lesions from ulcerative colitis (UC) and Crohn's disease (CD) patients. There was a significant positive correlation between IL-17 and C3 mRNA expression in the IBD mucosa. IL-17 stimulated a dose- and time-dependent increase in C3 mRNA expression and C3 secretion in colonic SEMFs. The C3 molecules secreted by colonic SEMFs were a 115-kDa alpha-chain linked to a 70-kDa beta-chain by disulphide bonds, which was identical to serum C3. The IL-17-induced C3 mRNA expression was blocked by p42/44 mitogen-activated protein kinase (MAPK) inhibitors (PD98059 and U0216) and a p38 MAPK inhibitor (SB203580). Furthermore, IL-17-induced C3 mRNA expression was inhibited by an adenovirus containing a stable mutant form of I kappaB alpha. C3 and IL-17 mRNA expressions are enhanced, with a strong correlation, in the inflamed mucosa of IBD patients. Part of these clinical findings was considered to be mediated by the colonic SEMF response to IL-17.

Strontium-90: concentrations in surface waters of the Atlantic Ocean.

From the large body of analyses of strontium-90 in surface waters of the Atlantic Ocean, annual average concentrations (from 10 degrees N to 70 degrees N) have been compared to those predicted. The data indicate higher fall-out over ocean than over land and confirm the rapid rates of down-mixing shown by most studies of subsurface strontium-90.

A case of polyarteritis nodosa with periurethralaseptic abscesses and testicular lesions.

We describe a 54-year-old man presenting with cutaneous ulcerations, livedo reticularis, numbness of the legs, and skin histological findings compatible with the diagnosis of polyarteritis nodosa (PAN). Initial treatment with 50 mg/day of prednisolone (PSL) was effective. However, the symptoms and signs recurred, and the patient developed multiple periurethral aseptic abscesses, urethra-cutaneous fistula, and testicular lesions after tapering of PSL therapy. The condition improved with PSL and cyclophosphamide administration. Since penile and testicular vasculitis could be associated with PAN, although rarely, we should carefully distinguish such an involvement from infection and malignancy.

Detection, characterization, and bioavailability of membrane-associated iron in the intact sickle red cell.

It is hypothesized that membrane-associated iron in the sickle red cell is of pathophysiologic importance, but the actual existence of such iron in the intact cell has been questioned. Using a strategy whereby membrane iron can be detected through its bioavailability for catalyzing peroxidation, we used phospholipid exchange protein to load membranes of intact erythrocytes (RBC) with approximately 2% phosphatidylethanolamine hydroperoxide (PEOOH) and monitored the development of peroxidation by-products during subsequent incubation. Normal RBC loaded with PEOOH developed very little peroxidation, but vitamin E-replete sickle RBC showed an exuberant peroxidation response that was not seen in cells loaded with control nonoxidized phosphatidylethanolamine. Ancillary studies of sickle RBC revealed that the catalytic iron included both heme iron and free iron located at the bilayer inner leaflet. Significantly, these studies also revealed that peroxidation after PEOOH loading is promoted by cellular dehydration and inhibited by hydration, thus identifying a dynamic interaction between hemoglobin (sickle >> normal) and membrane lipid. High-reticulocyte control RBC and sickle trait RBC behaved exactly like normal RBC, while HbCC RBC and RBC having membranes gilded with hemoglobin iron because of prior exposure to acetylphenylhydrazine showed an abnormal peroxidation response like that of sickle RBC. Indeed, the peroxidation response of RBC loaded with PEOOH paralleled amounts of iron measured on inside-out membranes prepared from them (r = 0.783, P < 0.01). These studies corroborate existence of membrane-associated heme and free iron in the intact sickle cell, and they document its bioavailability for participation in injurious peroxidative processes. That association of cytosolic sickle hemoglobin with membrane lipid is modulated by cell hydration status provides a mechanism that may help explain increased development of oxidative membrane lesions in abnormally dehydrated sickle RBC regardless of the mechanism underlying their formation.

Simulation of tissue properties in irreversible diffuse obstructive pulmonary syndromes. Enzyme digestion.

The length-tension properties of alveolar wall from normal cats were studied before and after exposure to enzymes naturally found in mammals (elastase, trypsin, collagenase, hyaluronidase). Hyaluronidase effected little change while all the proteolytic enzymes altered the mechanical properties of lung tissue. Collagenase removed the "mechanical stop" and the alveolar walls fractured at low forces. The properties of wall exposed to trypsin resembled those of elastase-treated tissue. Elastase increased the extension necessary to reach a given force and increased the maximum length (L(max)) and resting length (L(o)). Maximum extensibility (lambda(max)), the ratio of L(max) to L(o), fell with both elastase and trypsin digestion. A reduction in lambda(max) simulates the changes in alveolar wall properties seen in the lungs of the aged and in those with an irreversible diffuse obstructive pulmonary syndrome (DOPS(I)). Unlike these states, however, the energy loss in stretching alveolar wall increased with elastolysis. Furthermore, the changes in L(o) necessary to effect a change in lambda(max) of alveolar wall comparable to that seen in DOPS(I) were excessive. The altered tissue properties that occur in man with obstructive pulmonary syndromes could not be produced with these proteolytic enzymes or with hyaluronidase.

Simulation of lung tissue properties in age and irreversible obstructive syndromes using an aldehyde.

Weak solutions of CHOH alter tissue properties, probably by forming intermolecular cross-linkages. The maximum length (Lmax) to which alveolar wall can be extended is reduced. If exposed to CHOH while extended, the resting length (LO) of alveolar wall increases. Maximum extensibility (Lambdamax equal to Lmax/LO) decreases. Similar changes are found in the alveolar wall of man with aging and are significantly more marked in patients with irreversible obstructive pulmonary syndromes. A reduction in the energy loss of the length-tension cycle (hysteresis) was seen after exposure to CHOH, however, that does not occur with age or in obstructive syndromes. Because an exposure of alveolar wall to elastase increases LO and hysteresis, we used a staged exposure to CHOH followed by elastase. Tissue suitably prepared by exposure to CHOH followed by elastolysis better simulates the tissue changes of age and irreversible obstructive syndromes.

Radioactivity thresholds for sentinel node biopsy in breast cancer.

AIMS: The aim of the present study is to clarify the level of radioactive lymph node should be biopsied after the most radioactive SN is removed. METHODS: SNB using radionuclide was performed in our hospital for 1179 primary breast cancers between April 2000 and October 2005; most (1177/1179) were performed successfully. Our criterion for harvesting SNs is to remove tissue until no radioactive site is present. The level of radioactivity and the order of removal of each lymph node were compared with pathologic results. RESULTS: More than 2 (overall average 1.9) radioactive SNs were biopsied in 686 of 1177 breasts. Cancer positive results were recorded for 142 breasts with multiple SNs. In 142 breasts, 64 showed metastasis to the most radioactive node only, 39 showed metastasis other than the most radioactive node only, and 39 showed the most radioactive node and other radioactive nodes. Moreover, if several other criteria were applied, false-positive cases were increased significantly. CONCLUSIONS: It is necessary to harvest radioactive lymph nodes other than the most radioactive. Moreover, efforts to remove every radioactive lymph node will minimize false-negative results.

Effective esophageal balloon dilation for esophageal stenosis in recessive dystrophic epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited disorder of squamous epithelium that results in dystrophic scarring of the skin after minor trauma. RDEB is classified into two subtypes: Hallopeau-Siemens (HS) and non-Hallopeau-Siemens (nHS). Although severe scarring of the skin is the most common and obvious complication of the disease, esophageal scarring with formation of long strictures may also develop. Treatment options for esophageal stenosis in patients with RDEB include steroids, hyperalimentation, esophageal dilation and replacement. This report describes a child who was dilated immediately after diagnosis of severe esophageal stenosis subsequent to nHS-RDEB and managed successfully. Endoscopic esophageal balloon dilation under fluoroscopic control was very useful for detecting the region of stenosis and bougienage. The literature on such injuries is reviewed here, and the problems associated with the treatment of children with esophageal stenosis associated with RDEB are discussed.

Selective toxicity of MKT-077 to cancer cells is mediated by its binding to the hsp70 family protein mot-2 and reactivation of p53 function.

MKT-077, a cationic rhodacyanine dye analogue has been under preclinical cancer therapeutical trials because of its selective toxicity to cancer cells. Its cellular targets and mechanism of action remain poorly understood. Here we report that MKT-077 binds to an hsp70 family member, mortalin (mot-2), and abrogates its interactions with the tumor suppressor protein, p53. In cancer cells, but not in normal cells, MKT-077 induced release of wild-type p53 from cytoplasmically sequestered p53-mot-2 complexes and rescued its transcriptional activation function. Thus, MKT-077 may be particularly useful for therapy of cancers with wild-type p53.

Variation of microvascular blood flow augmentation--supercharge in esophageal and pharyngeal reconstruction.

AIM OF THE STUDY: A gastric tube is commonly used in thoracic esophageal reconstruction. When a gastric tube is not available, pedicled jejunum transfer and colonic interposition are alternative methods. Oral end of the reconstructed esophagus occasionally has poor blood flow and may result in partial necrosis of the oral segment. We performed additional microvascular blood flow augmentation, the "supercharge" technique, to improve a blood flow circulation in the oral segment of the reconstructed esophagus. METHODS: A series of 86 esophageal reconstructions with microvascular blood flow augmentation using the "supercharge" technique were performed. Reconstructive methods included a gastric tube in five patients, a gastric tube combined with a free jejunual graft in one, an elongated gastric tube in eight, a pedicled colonic interposition in 22, and a pedicled jejunum in 50. Recipient vessels were used in neck or chest region. RESULTS: The color and blood flow of the transferred intestine appeared greatly improved after microvascular blood flow augmentation. Thrombosis was noticed in three patients during the surgery, and all thrombosies were salvaged by re-anastomosis. There were only three patients with partial graft necrosis of oral segment, two patients with anastomotic leakage, one anastomotic stricture. CONCLUSIONS: Augmentation of microvascular blood flow by this "supercharge" technique can be expected to reduce the risk of leakage and partial necrosis of the transferred intestine. This technique contributes to the successful reconstruction of esophageal defect.

Gros1, a potential growth suppressor on chromosome 1: its identity to basement membrane-associated proteoglycan, leprecan.

By immunoscreening with an antibody raised against a plasma membrane protein, we have cloned a growth suppressor gene, Gros1 and assigned it to short arm of human chromosome 1. Two alternatively spliced forms of the gene encoding 84- and 41-kDa (carboxy-terminus truncated) proteins were cloned. The two transcripts, 4.4 and 2.7 kb, were expressed weakly in most of the human tissues, with a high expression of the smaller transcript in placenta, ovary and testis. Normal human fibroblasts in culture showed two transcripts, with a higher level of expression of the 4.4 kb transcript. Transformed cells on the other hand showed predominant expression of the 2.7 kb transcript. Two Gros1 transcripts were also detected in most of the mouse tissues. Stable transfection of the mouse cDNA encoding the 85-kDa protein into NIH3T3 cells resulted in their slow growth and reduced colony-forming efficiency. Stable clones expressing antisense RNA on the other hand exhibited higher colony forming efficiency. While our data implied that Gros1 is a novel growth suppressor gene on human chromosome 1, an independent study has recently characterized its rat-homolog as a leucine proline-enriched novel basement membrane-associated proteoglycan leprecan. We describe here cloning, expression and biological activity analysis implying that this novel proteoglycan is a potential growth suppressor on chromosome 1p31, frequently altered in many malignancies.

Phospholipid asymmetry during erythrocyte deformation: maintenance of the unit membrane.

To assess the red blood cell (RBC) membrane's ability to maintain normal phospholipid orientation in the face of deforming stress, we examined RBC subjected to elliptical, tank-treading deformation. As determined by accessibility to phospholipase digestion and by labelling with fluorescamine, normal RBC are able to fully preserve their phospholipid asymmetry despite attaining over 96% of their maximal possible deformation. Phospholipid orientation is unchanged during deformation even for RBC that are ATP-depleted or vanadate-treated and for RBC that already have destabilized phospholipids due to treatment with t-butyl hydroperoxide. These data indicate that maintenance of phospholipid organization during marked deforming stress and tank-treading motion of the membrane is ascribable predominantly to the passive stabilizing effect of membrane proteins. This provides additional evidence for the concept of a unit membrane characterized by intimate associations between lipid and protein.

Enhanced expression of multiple forms of VEGF is associated with spontaneous immortalization of murine fibroblasts.

The mechanism(s) involved in immortalization that constitute the first step during malignant transformation has been the subject of our interest. By the use of spontaneously immortalized mouse embryonic fibroblasts we have earlier identified two stages of immortalization which are characterized by growth characteristics of the cells, their conditioned medium and the protein markers such as p53, p81 and mortalin (Kaul et al. (1994) Biochim. Biophys. Acta, in press). The present study was planned to purify the mitogenic factors from the conditioned medium of stage II cells. Sequential purification by chromatography followed by peptide sequencing has characterized one of these as vascular endothelial growth factor (VEGF). Further analysis by RT-PCR suggests that the spontaneously immortalized stage II fibroblasts have enhanced synthesis and secretion of VEGF as compared to their mortal parent cells. Expression of a novel 304 bp long form of VEGF is identified in immortal fibroblasts in addition to the three known alternatively spliced forms. The study points to the involvement of VEGF function during spontaneous immortalization of mouse embryonic fibroblasts.

Self-adaptive modification of red-cell membrane lipids in lecithin: cholesterol acyltransferase deficiency. Lipid analysis and spin labeling.

In a patient with lecithin: cholesterol acyltransferase deficiency, free cholesterol was markedly increased, and esterified cholesterol was diminished. In the patient's plasma, an increase in phosphatidylcholine (PC) and a decrease in sphingomyelin were observed. Concomitantly, an increase in a shorter acyl chain 16:0 was noted in PC, sphingomyelin and phosphatidylethanolamine (PE). In contrast to these results, longer chains such as 22:0 and 24:0 were decreased, especially in sphingomyelin. Unsaturated double bonds such as 18:1 was also increased in PC and PE. In the red-cell membrane lipids, the increase in free cholesterol was counteracted by an increase in PC and by a decrease in sphingomyelin and PE, reflecting changes in the patient's plasma lipids. Increased 16:0 (in PC) and decreased 18:0 and 24:0 were observed. The increased plasma free cholesterol due to metabolic defect (lecithin: cholesterol acyltransferase deficiency) led to decreased red-cell membrane fluidity. This effect appeared to be counteracted by changing phospholipid composition (increased PC and decreased sphingomyelin and PE), by increasing shorter chains (16:0), by decreasing longer chains (18:0 and 24:0) and by increasing unsaturated double bonds (18:2). These results can be interpreted as a self-adaptive modification of lecithin: cholesterol acyltransferase deficiency-induced red-cell membrane abnormalities, to maintain normal membrane fluidity. This speculation was supported by the ESR spin-label studies on the patient's membrane lipids. The normal order parameters in intact red cells and in total lipid liposomes were decreased if cholesterol-depleted membrane liposomes were prepared. Thus, the hardening effect of cholesterol appeared to be counteracted by the softening effects described above. Overall membrane fluidity in intact red cells of the lecithin: cholesterol acyltransferase-deficient patient was maintained normally, judged by order parameters in ESR spin-label studies.

Identification of genetic events involved in early steps of immortalization of mouse fibroblasts.

The spontaneously immortalized early passaged fibroblasts from three different strains of mouse are observed to represent two distinct stages of immortalization. The cells at stage I are characterized by slow growth rate, contact inhibition and requisition of serum factors for their growth and proliferation. Stage II cells are marked by fast, multilayer growth that is independent of serum supplementation in growth medium and by the elevated levels of the two marker proteins, i.e., p53 and p81. The change from cytosolic distribution of mortalin, a senescence inducing protein (J. Biol. Chem. (1993) 268, 6615-6621; 22239-22242) to the perinuclear locale is detected as an early event during cellular immortalization. Furthermore, the distinct stages could be characterized by thermal analysis of intact cells, that to the best of our knowledge is employed for the first time for the analysis of cellular mortal and immortal phenotypes. The study characterizes at least two distinct end points in rodent transformation suggesting that there are multiple routes to immortalization.

Preferential impairment of nitric oxide-mediated endothelium-dependent relaxation in human cervical arteries after irradiation.

BACKGROUND: Vascular abnormalities are a major cause of postoperative complications in irradiated tissues. Endothelial cell dysfunction characterized by diminished endothelium-dependent relaxation may be involved. We examined the endothelium-dependent relaxation and morphology of the endothelium in irradiated human cervical arteries. METHODS AND RESULTS: Irradiated arteries were taken from the neck region of patients who had radiation therapy. Arteries from patients who did not receive radiation therapy were used as controls. Endothelium-dependent relaxation to acetylcholine and A23187 was impaired in irradiated arteries. Norepinephrine-induced contraction and sodium nitroprusside-induced relaxation were unchanged. In control arteries, N(omega)-nitro-L-arginine and indomethacin each caused a partial inhibition of endothelium-dependent relaxation. In irradiated arteries, the impaired endothelium-dependent relaxation was unaffected by these agents, but it was abolished by high K(+). Acetylcholine produced similar degrees of hyperpolarization in control and irradiated arteries. Immunohistochemical examination for endothelial nitric oxide synthase indicated no expression in the endothelium of irradiated arteries. Electron scanning microscopy showed morphologically intact endothelial cells in irradiated arteries. CONCLUSIONS: In irradiated human cervical arteries, the nitric oxide- and prostacyclin-mediated endothelium-dependent relaxation, but not endothelium-derived hyperpolarizing factor-mediated relaxation, are specifically impaired, without significant morphological damage of the endothelium. The impaired nitric oxide-mediated relaxation was associated with a lack of endothelial nitric oxide synthase expression. Our results suggest the importance of impaired endothelial function in irradiated human blood vessels, which may partly explain the development of vascular stenosis and poor surgical wound healing in irradiated tissues.

Evaluation of covered metallic stents in malignant biliary stenosis--prominent effectiveness in gallbladder carcinoma.

BACKGROUND/AIMS: The survival time of patients with unresectable malignant biliary stenosis and the patent period of metallic biliary stents are different in each disease. The efficacy of the covered metallic stent was analyzed according to the primary disease. METHODOLOGY: Seventy-three patients with bile duct carcinoma (12 cases), gallbladder carcinoma (22 cases), and pancreas carcinoma (39 cases) were retrospectively enrolled. Covered metallic stents were used in 42 patients and uncovered metallic stents in 31 patients. The patency of covered stents was compared with that of uncovered stents for each disease. RESULTS: The patent rate at 6 months after insertion was 80.6% (95% CI [72.6%, 88.6%]) for the covered stent, and 49.5% (95% CI [37.6%, 61.4%]) for the uncovered stent. The mean patent periods of the covered stent and the uncovered stent were 14.6 and 27.6 months for bile duct carcinoma (p=0.424), 12.7 and 3.0 months for gallbladder carcinoma (p=0.003), and 11.9 and 9.6 months for pancreas carcinoma (p=0.919), respectively. CONCLUSIONS: The covered metallic stent was the most effective in patients with gallbladder carcinoma.

Late expression of red cell membrane protein 4.2 in normal human erythroid maturation with seven isoforms of the protein 4.2 gene.

The expression of protein 4.2 in normal human erythroid cells was studied utilizing erythroblasts from bone marrow and erythroid cells cultured by the two-phase liquid culture method from burst-forming unit erythroid (BFU-E) in peripheral blood. As opposed to spectrin, which was expressed in erythroid progenitors or very early erythroblasts, protein 4.2 was first detected in late erythroblasts with a morphology nearly identical to orthochromatic erythroblasts. Among the various major membrane proteins, the expression of protein 4.2 was the latest. At the gene level, protein 4.2 gene mRNA was expressed in early erythroblasts. During normal erythroid maturation, the expression of seven different protein 4.2 gene products was observed by Southern blot analysis. These seven gene products appeared to be derived from protein 4.2 gene in the presence or absence of skipping of the 90 bp in exon 1, exon 3, and/or exon 5, as judged by deduction from the protein 4.2 sequence. Therefore, it can be speculated that protein 4.2 is expressed after the cytoskeletal network has been constructed and assembled with integral proteins in the membrane lipid bilayer.

Expression of proto-oncogenes in normal and tumor tissues of human skin.

The expression of c-fos, c-myc, Ha-ras, N-ras, EGF-receptor, and cardiac actin genes was examined in 7 normal epidermis, 3 cellular nevi, and 8 skin tumors including 6 malignant and 2 benign tumors of human origin. These genes were transcribed in most normal and tumor tissues, though no tumor-specific expression of proto-oncogenes (c-fos, c-myc, Ha-ras, and N-ras) could be detected. However, there was a characteristic parallelism between the expression of c-fos and c-myc in normal epidermis, while the parallelism was not always definite in skin tumors. The ratio of c-fos/c-myc transcripts in normal epidermis was constant compared with the expression of other genes examined. These data suggest that c-fos and c-myc are expressed in all normal skin tissues, and that maintenance of a constant ratio of c-fos/c-myc is closely related to ordered cell growth of the tissues.